There is a crucial need for the development of new strategies that would achieve transplantation tolerance without the deleterious side effects of drug-mediated immunosuppression. Assessing potential alternatives, we have established that the transfer of graft-type MHC class II gene(s) in recipients promotes spreading T cell tolerance to subsequent kidney grafts; i.e., the absence of host T cell response to all graft-associated major and minor antigens. Our long-term goal is to elucidate the mechanism of class ll-mediated tolerance as a prerequisite to the development of therapeutic protocols for transplantation tolerance. The specific hypothesis is that class II peptides, derived from the transferred gene and the class ll-matched graft, are the key inducers of tolerance through the activation of host regulatory T cells (T-regs). This hypothesis is based on the observations that: 1) transferred CI2 molecules are converted into peptides in antigen-presenting cells (ARC); 2) CI2 transgene-derived peptides activate T-regs in vitro for suppression of T effector cell proliferation and 3) the injection of T-regs, but not T effector cells, isolated from graft acceptors convey tolerance to identical grafts implanted in immunocompetent hosts. These observations have defined the focus of the project which is on the CI2-induced, T-reg-mediated, regulatory tolerance to CI2-matched grafts. The specific aims are to: 1. Evaluate the tissue-distribution patterns of the CI2 transgene expression and its effects on the immune system of the recipient. We will correlate the pattern of transgene expression in graft hosts with its outcome on graft survival and immune responses to donor antigens. 2. Analyze the impact of donor CI2 surface expression versus peptide expression on host immune status and tolerance to CI2-matched grafts. Using vectors for directed class II expression as surface molecules or peptides on APCs and non-professional APCs, we will ascertain in vivo whether class II peptide expression on APCs is a requirement for spreading tolerance. 3. Study the specificity of T-reg activation and suppression on alloreactive effector T cells. We will: (i) characterize the class ll-derived peptides that induce T-reg activation; (ii) use peptide-activated T-regs in adoptive transfer experiments; and, (iii) assess whether class II antigens from the graft are involved in T-reg activation and spreading tolerance.